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‘Caged cytoskeletons’: a rapid method for the isolation of microtubule‐associated proteins from synchronized plant suspension cells
Author(s) -
McCutcheon S.,
Hemsley R. J.,
Jopson M. F.,
Lloyd C. W.
Publication year - 2001
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2001.01134.x
Subject(s) - microtubule , mitosis , cytoskeleton , tubulin , microbiology and biotechnology , depolymerization , cellulase , biology , chemistry , cell , biochemistry , enzyme , organic chemistry
Summary In the cytoskeleton method for isolating microtubule‐associated proteins MAP65, DcKRP120‐1 and DcKRP120‐2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti‐microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY‐2 cells for only several minutes allows extraction of detergent‐soluble proteins, leaving synchronized ‘caged cytoskeletons’ for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti‐microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin‐related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.